As much as we don’t want to admit it, miscarriages do happen. They are traumatic experiences that most patients would rather not have to think about, as the prospect seems almost too frightening to consider. Many factors contribute to miscarriages; some patients also experience early pregnancy loss – although similar, it is not the same thing as a miscarriage and is caused by different factors.
To help us sort all of this out, we invited Dr Robert Najdecki, co-founder of the Assisting Nature clinic in Thessaloniki, Greece. In his presentation, he explained the differences between miscarriages and implantation failures and how modern reproductive techniques, such as PGT-A, may help. He was joined by clinical embryologist Tatiana Chartomatsidou.
The rapid development of reproductive science naturally leads to a deeper understanding of reproductive processes. As such, we now distinguish different types of pregnancy loss – miscarriages, early pregnancy loss, implantation failures, stillbirths, et cetera.
Miscarriages are defined as the natural death of an embryo or foetus. It occurs in 30% of all conception and 10% of all clinical pregnancies. The term “recurrent miscarriage” means at least two consecutive pregnancies ended prematurely; this affects between 1% and 3% of all women. Miscarriages can be caused by a myriad of reasons, such as:
- Uterine anomalies,
- Endocrine reasons,
- Immunologic reasons,
- Infectious causes,
- Genetic reasons, such as embryo aneuploidy
Although related to miscarriage, implantation failure is a separate phenomenon. Whether or not the embryo implants depends on many factors, such as the endometrial environment, embryo quality and chromosomal health, and others. Embryo implantation failures are most commonly caused by a decrease in endometrial receptivity due to uterine abnormalities, endometriosis, immunological issues and embryo aneuploidy. The term “implantation failure” is used to describe patients who have never shown an increased level of hCG (human chorionic gonadotropin), or those who don’t exhibit any evidence of a gestational sac on ultrasound scans. It applies to both patients undergoing assisted reproductive treatments, as well as those trying to conceive naturally. The term “repeated implantation failure” describes numerous failed embryo transfers performed using assisted reproductive technology (ART).
The process by which the endometrium changes itself in preparation for, and during, pregnancy is called decidualisation. The endometrial tissue transforms itself into morphologically and functionally distinct decidua. It occurs after ovulation in the menstrual cycle and continues further if an embryo successfully implants itself.
The sensitivity of the uterus to implantation is divided into three phases: pre-receptive, receptive, and post-receptive. As the name suggests, the receptive phase is the only phase in which embryos successfully implant, as certain characteristics required for implantation are only present during that phase. The endometrium plays an important role in embryo selection, as the decidual cells can recognize impaired embryos and inhibit implantation. Cells which have not undergone decidualisation can’t express such a response. The ability to undergo decidualisation is impaired in women suffering from recurrent implantation failure. As such, the result of implantation relies heavily on effective cross-talk between the embryo and a decidualised endometrium. If the endometrium fails to select a proper embryo – this situation being called, obviously, an endometrial selection failure – an early pregnancy with a significant risk of failure later on may occur.
Embryos with an abnormal number of chromosomes are considered aneuploid. Normally, an embryo has 46 chromosomes – 23 from each parent. Sometimes, however, due to DNA fragmentation or other defects, embryos can gain chromosomes and form trisomies, or lose chromosomes, forming monosomies.
There are two distinct kinds of aneuploidies – “whole chromosome” aneuploidies, in which an entire chromosome is missing, or “structural” aneuploidy (sometimes called “segmental”), in which chromosomal regions are rearranged.
Aneuploidy is the most significant single factor affecting early pregnancy loss and miscarriage. 65% of abnormal embryos end in spontaneous miscarriages. Other complications include implantation failure or congenital disabilities if a child is born.
Aneuploidy can occur in both embryos and gametes. The most common causes of aneuploidy is advanced maternal age; the older the mother, the higher chance of her oocytes being defective. The rate of aneuploidy in eggs increases significantly after the age of 35; it may reach levels as high as 70% or 80% by the age of 45. This increased aneuploidy rate, in turn, leads to increased likelihood of implantation failure, higher miscarriage rates and a lower chance of a healthy live birth rate.
Poor sperm quality can also contribute to aneuploidy. Other causes of aneuploidy in embryos include hormonal overstimulation in IVF cycles, the presence of chromosomal abnormalities in either of the parents and chromosomal errors in embryo cell division.
The most common trisomies are trisomy 21 (Down syndrome), 18 (Edwards’ syndrome), 13 (Patau syndrome), and 47 (Klinefelter syndrome, also known as XXY). The most common monosomy is 45, or Turner syndrome.
IVF labs perform assessments on embryos in order to prevent the transfer of aneuploid embryos; these assessments, however, are based on morphological criteria. The number of blastomeres within the embryo is recorded on day three, along with the fragmentation rate. On day five, the embryos undergo another assessment in order to determine which ones have reached the blastocyst stage; subsequently, the blastocysts’ morphology is assessed. However, morphology alone does not tell us if the embryos are chromosomally normal; around 44.9% of embryos exhibiting good morphological traits are revealed to be aneuploid after genetic testing.
Implantation rates present a strong argument for genetic testing. The implantation rates for embryos which have been genetically proven to be euploid remain at a stable level – almost 50% – with the age of the patient having no effect. Conversely, implantation rates for embryos which do not undergo testing drop steadily with the age of the patient.
Genetic testing takes many forms. Previously, the terms Preimplantation Genetic Screening (PGS) and Preimplantation Genetic Diagnosis (PGD) have been used to describe the two main types of testing. Nowadays, the umbrella term Preimplantation Genetic Testing (PGT) is used to avoid confusion. PGT is further subdivided into categories:
- PGT-A: Preimplantation Genetic Testing for aneuploidies,
- PGT-SR: Preimplantation Genetic Testing for chromosomal structural rearrangements,
- PGT-M: Preimplantation Genetic Testing for monogenic diseases, such as cystic fibrosis
Out of the three categories, PGT-A is the one enjoying the most popularity. It is a genetic test that can be performed on day three and day five embryos. On day three, the embryo is in the cleveage stage – it consists of eight cells, of which one is removed for testing in a genetics laboratory. On day five, during the blastocyst stage, the embryo is comprised of over 200 cells, allowing embryologists to collect a bigger sample. The cells are taken from the trophectoderm – the part of the embryo which will become a placenta.
The methods used in genetic testing are developing rapidly. Next Generation Sequencing (NGS) is a new technique which boasts an impressive 99.98% consistency rate for its results. It provides a greater scope of information to geneticists, it reveals mosaicism within the embryos, as well as minimising the risk of receiving false positive or negative results. It’s a flexible and cost-effective technique, which makes it applicable in many different scenarios, even as a routine test. The only downside is that it takes about twenty days for results to be generated.
Despite its popularity, or perhaps because of it, many myths have sprung up around PGT-A, chief among them being the claim that PGT-A improves the reproductive potential of embryos, or that it somehow “repairs” them. This couldn’t be farther from the truth – all PGT-A is, is a diagnostic test. The only thing it does – and the only thing it’s intended to do – is help embryologists select the best embryo to be transferred. The higher implantation rates, lower chances of miscarriages and shorter time to pregnancy are simply a side effect of choosing the right embryo.
PGT-A is not perfect, either. It can’t detect embryos which are affected by mosaicism in less than 20% of its cells; rarely, some embryos don’t give conclusive results. And, of course, it can’t do anything about cases where a euploid embryo simply isn’t generated. And then there’s the matter of sex selection.
PGT-A can reveal the sex of the embryo, although making a decision based on that information is illegal under Greek law (as well as in most other European countries). As such, the information about the embryo’s sex is not included in the test results. The only exception is if there’s a documented risk of the child suffering from a genetic disease linked to sex chromosomes.
We touched on mosaicism briefly – mosaic embryos are those which contain both euploid and aneuploid cell lines. They make up about 21% of all blastocysts and can be categorised as either transferrable or non-transferrable; this distinction is made based on the level of mosaicism and which chromosomes are involved. Although they can result in normal and viable pregnancy, there are only used in case there is no euploid embryo available for transfer, as they carry a higher risk of miscarriage and implant at a lower rate compared to genetically normal embryos.
The process of PGT-A testing is very simple. The patient undergoes controlled ovarian stimulation, as usual; oocytes are retrieved and fertilised using the ICSI procedure, after which the newly created embryos are allowed to develop for five days. On day five, the biopsy is performed and the embryos are frozen until the results are known – a period of about three weeks.
Once the results are available, only the euploid embryos can be thawed and transferred. If there are more than one, the surplus embryos remain in storage for future treatments; aneuploid embryos cannot be stored in an embryo bank or transferred – they either get destroyed or donated for research, depending on the patient’s wishes.
Patients are commonly concerned about the biopsy aspect of PGT-A. After all, it is an invasive procedure which interferes with the embryo’s cellular makeup. Dr Najdecki and Ms Chartomatsidou assure us, however, that it is a safe procedure – although each IVF clinic has their own biopsy technique, only highly trained and experienced staff are allowed to carry out the procedure, minimising the possibility of any adverse effects almost completely. Furthermore, only trophectoderm cells are taken – these are the cells that later form the placenta, not the foetus; those cells remain intact. The vitrification process used to store the embryos until the test results come back is also extremely effective, with a survival rate of up to 98%.
So, who exactly should consider PGT-A testing? Women of an advance maternal age are prime candidates for testing, as are couples with severe male factor infertility, as egg and sperm quality have a major impact on the health of the embryos. Couples suffering from recurrent miscarriages, recurrent implantation failures and those with a known chromosomal abnormality should also consider having their embryos tested. PGT-A is also available in donation treatments. Embryos created from donor gametes aren’t guaranteed to be aneuploid; in fact, aneuploid embryos still appear in donation cycles, just at a lower rate.
The treatment itself is just one piece of the larger puzzle, however. Counselling is also extremely important, both before and after the treatment. Before undergoing any treatments, patients should assess their reproductive history, determine whether there are any parental chromosomal abnormalities, undergo karyotyping – simply put, determine whether or not there are indications for PGT-A, as well as understanding associated risks and other options available, such as egg donation. Following the treatment, counselling should focus on the interpretation of the results and developing a personalised strategy for the patient, as well as discussing the options in case no euploid embryo was found.
The advantages of PGT-A seem to speak for themselves: increased implantation rates, decreased clinical pregnancy loss rates, higher chance of a live birth, shorter time to pregnancy… By simply selecting the right embryo to transfer, couples undergo less stress – failed transfers and pregnancy loss is less likely, after all. Previous IVF failures can also be explained by PGT-A, encouraging patients to continue treatment. For those with recurrent aneuploid loss, PGT-A may help determine if there are any therapeutic options to look into.
According to statistical data collected in-house at Assisting Nature, pregnancy rates are high across the board for those women who undergo PGT-A, and have euploid embryos available to transfer. In patients aged 35 to 39, the pregnancy rates reach a level of around 70%. 65% of patients in that age group achieve a clinical pregnancy. The live birth rate for those patients is around 60%. Although these rates are affected by the patient’s age, the results are still higher than for those patients who do not opt for PGT-A testing.
To undergo an embryo transfer, however, patients must have a euploid embryo. Out of all couples who undergo testing, 54% of them end up with no euploid embryos available for transfer. In egg donation cycles, however, this problem is mitigated. 100% of the patients undergoing egg donation treatments at Assisting Nature had euploid embryos available for transfer, with a pregnancy rate higher than 80% and a live birth rate higher than 75%.
In conclusion, PGT-A, despite being an invasive technique requiring extreme precision – and a highly trained embryologist – provides precise genetic information about the embryo, while also helping to increase pregnancy and live birth rates, and decrease miscarriage rates. All of this without hurting the embryo. Dr Najdecki and Ms Chartomatsidou express a hope that in the future, it becomes a standard part of IVF treatments.
Miscarriages and embryo implantation failures vs PGT-A – Questions & Answers
Is it possible to thaw a frozen embryo for PGT and freeze it later (after cell removal) for later transfer? Does the viability of the embryo decrease as a result of this?
Yes, it’s possible. The golden standard, however, is to fertilise egg cells into embryos and perform genetic testing on day 5 of their development – and then freeze them. This method provides the best results. Thawing and freezing an embryo, while possible, can skew the results of PGT testing, which is why we test embryos that way if there is no other way.
Are frozen egg cells of the same quality as fresh ones? Are fertilisation and implantation rates of fresh and cryopreserved eggs comparable?
There is a small difference; as a rule, we prefer using fresh eggs. However, thanks to new vitrification techniques and time-lapse incubators, the results we get from using frozen eggs are very close to those we get from fresh ones.
Is PGT the same as PGS and PGD?
PGS is the old name for the technique we now call PGT-A. There is also PGT-R, which also screens for aneuploidies, but in much smaller regions of chromosomes. PGD is another thing entirely; it stands for Pre-Implantation Genetic Diagnosis. Nowadays, it’s called PGT-M, because what it allows us to do is detect monogenic diseases – that is, diseases caused by a single gene.
Is there any work being done to determine the success rate of varying types of mosaicism?
Like we said before, we usually only transfer mosaic embryos as a last resort; not all embryos afflicted by mosaicism are transferrable. Whether or not we decide it’s viable depends on the percentage of mosaicism within the embryo, as well as what kind of abnormalities are implied. This is also a personalised issue with most patients, as we need an opinion of a geneticist in order to determine if a mosaic embryo can be transferred.
Because PGT is such a new technique, how do we know it doesn’t carry risks that could manifest later in the child’s life?
While PGT is an invasive technique, it is always performed by highly experienced embryologists. Because of this, there is basically no harm done to the embryo. Studies are still, of course, trying to determine if there are any effects of PGT testing. Because PGT testing uses cells from the outer embryo in the blastocyst stage – that is, not the cells that become the foetus – the risk of any damage is minimal. In fact, we would go as far as wagering that in five years, every IVF cycle will be done with PGT-A testing, as it provides us with a lot of information in case the transfer fails or the pregnancy ends with a miscarriage.
Is there a relationship between egg quality and stimulation level?
There is a relationship between the quality of the egg and the stimulation process. However, the precise impact of this relationship is age-dependant – younger patients, for instance, are not affected by it at all. In some older patients, however, egg quality may be adversely affected by large amounts of gonadotropins used in the stimulation process – which is why those patients are put on a personalised hormone regimen.
What do you think of the new research that is being done on the fluid around the embryo rather than actual cells for PGT testing?
If the method turns out to be viable, it would be good news for all of us, as we’d have a non-invasive method of testing. It’s a very new idea and it remains to be seen if it’s something that could change PGT.
If it turns out testing embryo culture fluid delivers results as accurate and reliable as a biopsy, it would mean a revolution on the scale of NIPT – non-invasive prenatal testing. Like we said, however, this is all in the future – for now, we know too little about the technique to introduce it to embryology labs.
Do you anticipate PGT-A becoming a standard procedure in IVF?
We have seen that PGT-A not only reduces the average length of treatment, it also increases all of the IVF success rates – clinical pregnancy rates, live birth rates, et cetera. It could be a standard part of IVF in the future, if for no other reason than to help clinics boast about their high success rates. However, currently there is a cost associated with testing, which prevents PGT-A from becoming a standard procedure.
Currently, we’re developing new protocols in which we strongly recommend PGT-A in case the first embryo transfer attempt fails.
What are the main reasons for implantation failure with euploid embryos?
There are many other factors involved in the process of implantation. Implantation failure can be caused by small anatomical variations in the uterus, endometrial stimulation, et cetera… Unfortunately, the human body is not a machine; in-depth testing is required to pinpoint the exact fault.
Does genetic testing create “designer babies”?
There is a lot of discussion whether or not genetic testing in IVF could lead to a “world of eugenics”. In my opinion, PGT-A is a clear and ethical procedure; in fact, knowingly creating embryos and pregnancies with chromosomal abnormalities is unethical and unacceptable. This isn’t eugenics or “baby designing”.
Do you think PGT can be valuable in IVF programs with donor eggs for patients who experienced implantation problems in the past?
Yes. We advise our patients – if they are able to afford it, as it is an additional expense – to try PGT-A in donation cycles; even though our donors are all young and meet strict requirements, it’s still worth knowing whether the embryos are all euploid.
Why do some aneuploid embryos implant and then miscarry? Why do they not all fail to implant?
Some non-euploid embryos implant because they carry a chromosomal abnormality that may not be an issue during the implantation process. That same abnormality, however, may cause problems later, leading to a miscarriage. It all depends on which chromosome is affected.
What happens to aneuploid embryos?
Aneuploid embryos can’t be transferred, as they will not result in safe pregnancies. Embryos that are tested are stored until we receive the test results. Once we know which ones are aneuploid, the patient can decide whether the embryos get destroyed or donated for research.
Can a couple store embryos with aneuploidies at an embryo bank?
No. The only possibilities for aneuploid embryos are either disposal or donating them for research or education of new embryologists. The decision lies with the patients, but it must be one of these three options.